The main objective of this proposal is to understand better the association of actin with the human erythrocyte membrane through characterization and isolation of the actin binding protein. Because earlier studies have suggestd tat mechanochemical activities in the erythrocyte might involve actin, we have developed a method for purifying erythrocyte membrane actin, which is in every way tested like human muscle actin. For this actin to fulfill a mechanochemical role in the erythrocyte, we feel it should be attached to an integral membrane protein. Since actin can be solubilized easily from the erythrocyte membrane, we plan to use that actin-depleted membrane and purified actin to study the conditions required for actin to bind specifically to the membranes. The number and distribution of bound actin filaments will be followed under a number of ionic conditions and in the presence of agents such as certain hormones and antibodies. These agents affect the mechanochemical properties of the whole erythrocyte apparently through transmembrane effects of exernal proteins to which they bind on the submembrane structure of the erythrocyte. If the initial experiments indicate that the binding protein is present, we will attempt to isolate antibodies to the binding site protein as a tool for continuing studies. The antiserum, when extensively characterized to establish that it binds to only the one protein, can be used to determine the quantity and location of actin binding sites in the erythrocyte and related cells. The techniques of ultrathin frozen-sectioning and ferritin antibody staining would be used to visualize the binding sites in the electron microscope. We hope this study would be a basis for further investigation of actin-membrane associations. The understanding of the factors governing the binding of actin to membranes is necessary for the further understanding of control mechanochemical functions in non-muscle cells.